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研究生：	江國銘 Chiang, Kuo-Ming
論文名稱：	由導致遺傳性易感染分枝桿菌突變基因研究丙型干擾素受體的新奇分辨性訊息傳遞 Novel Differential Signal Transduction by IFN-γ Receptor by Disease-causing Mutation of Mendelian Susceptibility to Mycobacterial Diseases (MSMD)
指導教授：	謝奇璋 Shieh, Chi-Chang
學位類別：	碩士 Master
系所名稱：	醫學院 - 臨床醫學研究所 Institute of Clinical Medicine
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	58
中文關鍵詞：	遺傳性易感染分枝桿菌疾病、丙型干擾素接受體1 、丙型干擾素、菸醯胺腺嘌呤二核苷酸磷酸氧化酶
外文關鍵詞：	MSMD, IFNGR1, IFN-γ, NADPH oxidase priming effect, oxygen species
相關次數：	點閱：162 下載：2
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孟德爾氏遺傳性分枝桿菌易感染性疾病 (MSMD)是一種病患容易受各種非結核分枝桿菌(包括卡介苗)感染的罕見疾病。該疾病主要是體內丙型干擾素接受體異常，以及其他在介白素-12及丙型干擾素訊息途徑相關基因缺失所造成。丙型干擾素對於清除胞內感染病原體相當重要，其中包含活化菸醯胺腺嘌呤二核苷酸磷酸氧化酶 (NADPH oxidase)產生活性氧分子 (ROS)。我們在孟德爾氏遺傳性分枝桿菌易感染性疾病患者家族中，發現其丙型干擾素接受體1基因在第 818位上缺失四個核甘酸，造成丙型干擾素接受體1位於膜內蛋白質結構缺失，無法正常傳遞受丙型干擾素所調控的JAK-STAT1訊息而引發體染色體顯性遺傳的疾病。我們也發現丙型干擾素接受體突變的白血球喪失對丙型干擾素刺激產生IL-12的反應。然而，以丙型干擾素刺激來自病患的周邊血單核球細胞所引發起NADPH oxidase活化的反應並不受影響。因此我們推測即使缺失丙型干擾素接受體1膜內蛋白質的胜肽區域 (domain)，丙型干擾素接受體仍可以藉由丙型干擾素接受體2傳遞訊息。在本研究中，我們利用基因選殖技術將丙型干擾素接受體1基因及缺失膜內蛋白質的丙型干擾素接受體1 基因接入病毒載體並轉入293T細胞中。利用流式細胞儀，我們發現到相較於正常丙型干擾素接受體1，缺失膜內蛋白質的丙型干擾素接受體1會大量聚集在細胞膜上。進一步利用西方墨點法也發現到，在丙型干擾素刺激下，缺失膜內蛋白質的丙型干擾素接受體1 無法傳遞調控STAT1、p38 MAPK、Akt磷酸化途徑，但是仍然會誘導Erk1/2及p47phox磷酸化。此外，我們也發現到以丙型干擾素刺激缺失膜內蛋白質的丙型干擾素接受體1可以經由Erk1/2訊息途徑引發起菸醯胺腺嘌呤二核苷酸磷酸氧化酶的活化。這些結果顯示，在缺失膜內蛋白質的丙型干擾素接受體1中，丙型干擾素仍然可以傳遞訊息並調控細胞功能。

Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare congenital disorder with impaired immunity against mycobacterial pathogens. MSMD is caused by genetic defects in the IL-12/23-IFNγ circuit. IFN-γ immunity is important for control of intracellular organisms such as bacillus Calmette-Guérin (BCG) and other non-tuberculosis mycobacterium (NTM). With the immune response against the infection of intracellular organisms, the NADPH oxidase-produced oxygen species (ROS) play an important role. In our previous results, we found 3 MSMD patients from a family with IFNGR1 gene mutation (818del4) which leads to a truncated IFNGR1 lacking the entire cytoplasmic signal-transducing domain. This mutation leads to autosome domain (AD) IFN-γ receptor 1 (IFNGR1) deficiency in gene carriers. IFN-γ-induced IL-12 secretion from peripheral blood mononuclear cells (PBMCs) was deficient in patients with AD IFNGR1 deficiency. However, we found that IFN-γ-induced NADPH oxidase priming effect is normal in patient with AD IFNGR1 deficiency. These results implicated that parallel signals may be initiated by IFN-γ stimulation in both IFNGR1-dependent and IFNGR1-independent manners. In this study we cloned the WT and 818del4 mutant IFNGR1 DNA sequences into lentiviral expression vector and transfected the constructs into human embryonic kidney 293T cells. We detected a high surface expression level of IFNGR1 in 818del4 mutant IFNGR1-transfected cells. In addition, in 818del4 mutant IFNGR1-expressing cells, IFN-γ could not transmit the STAT1, p38 MAPK and Akt signaling, but could induce phosphorylation of Erk1/2 and phosphorylation of p47phox. Moreover, IFN-γ-induced phosphorylation of p47phox was inhibited by ERK1/2 inhibitor. In conclusion, the cytoplasmic truncated IFNGR1 can transmit differential signals for activation of NADPH oxidase after treatment with IFN-γ.

摘要..........................................I
Abstract……………………………………………………………………………………II
誌謝…………………………………………………………………….........................III
Abbreviation Index………………………………………………………………………VIII
Introduction	1
1 Interferon gamma (IFN-γ)	2
1.1 The IFN-γ receptor	3
1.2 Canonical IFN-γ signaling pathway	4
1.3 STAT1-dependent but JAK1-independent signaling	5
1.4 STAT1-independent signaling	6
2 IFNGR1 818del4 mutation causing MSMD	6
3 The NADPH oxidase	7
3.1 Priming of NADPH oxidase	9
3.2 The mechanism of priming of NADPH oxidase………………………….10
4 Research objective	11
Materials and Methods………………………………………………………………….12
1 Cell culture and cell stimulation	13
2 Amplified Wild type IFNGR1 and IFNGR1 (818del4) sequences	13
2.1 Reverse transcription-polymerase chain reaction (RT-PCR)	13
2.2 Polymerase chain reaction (PCR)………………………………………....14
2.3 Gene transfer	14
3 Transfected vector into cells……………………………………………………...15
4 Flow cytometry…………………………………………………………………..15
5 Cell sorting……………………………………………………………………….16
6 Western blotting………………………………………………………………….16
7 Antibodies………………………………………………………………………..17
8 Peripheral blood mononuclear cells (PBMCs) stimulated with IFN-γ………….17
9 ROS production…………………………………………………………………17
10 Statistical analysis……………………………………………………………...18
Results………………………………………………………………………………….19
1 Establishment of 818del4 mutant IFNGR1-expressing cells……………………20
1.1 Constructing the wild-type IFNGR1 and mutant IFNGR1 (818del4) sequences and analyzing the cell surface expression of IFNGR1………………20
1.2 Controlling the equal cell surface expression of IFNGR1 on WT and 818del4 mutant IFNGR1-transfected 293T cells……………………………….21
1.3 The cell surface expression level of IFNGR2 showed no difference between WT and 818del4 mutant IFNGR1-expressing 293T cells………………………21
1.4 IFN-γ enhanced the internalization of WT IFNGR1, but not in 818del4 mutant IFNGR1……………………………………………………………........21
2 IFN-γ induced phosphorylation of STAT1 in WT IFNGR1-expressing cells but not in 818del4 IFNGR1 mutant cells……………………………………………………..22
3 IFN-γ-induced inhibition of phosphorylation of Akt was dependent on IFNGR1………………………………………………………………………………23
3.1 IFN-γ inhibited the phosphorylation of Akt in WT IFNGR1-expressing cells but not in 818del4 IFNGR1-expressing cells……………………………...23
3.2 The phosphorylation of Akt was inhibited by IFN-γ in peripheral blood mononuclear cells (PBMCs) from healthy control but not in 818del4 IFNGR1 mutant PBMCs………………………………………………………………….24
4 IFN-γ induced phosphorylation of p38 MAPK in WT IFNGR1-expressing cells but not in 818del4 mutant IFNGR1-expressing cells………………………………...24
5 IFN-γ induced the phosphorylation of ERK1/2 in WT and 818del4 mutant IFNGR1-expressing cells…………………………………………………………….25
6 Phosphorylation of p47phox was induced by IFN-γ in 818del4 mutant IFNGR1-expressing cells……………………………………………………………26
6.1 IFN-γ induced phosphorylation of p47phox in 818del4 mutant IFNGR1-expressing cells through ERK1/2 pathways…………………………26
Discussions……………………………………………………………………………..28
1 The role of cytokine receptors in leukocyte signaling transduction and different cellular functions……………………………………………………………………..29
2 IFN-γ mediates the cellular function through different pathway………………...31
3 The regulation of IFN-γ in mycobacterial infections…………………………….32
4 Cross interaction between type I and type II interferon signaling pathway……..33
5 Future directions…………………………………………………………………34
References………………………………………………………………………………36
Table……………………………………………………………………………………43
Table 1: The signal pathways involved in the cellular response to IFN-γ……………44
Figures and legends…………………………………………………………………….45
Figure 1……………………………………………………………………………….46
818del4 mutant IFNGR1 was constructed and highly expressed on cell surface after transfection…………………………………………………………………………..48
Figure 2..……………………………………………………………………………...49
Phosphorylation of STAT1 was not induced in 818del4 mutant IFNGR1-transfected cells after IFN-γ stimulation	..50
Figure 3...……………………………………………………………………………..51
IFN-γ inhibited the phosphorylation of Akt in WT IFNGR1-expressing cells, but not in 818del4 mutant IFNGR1-expressing cells………………………………………..52
Figure 4……………………………………………………………………………...53
IFN-γ could not up-regulate the phosphorylation of p38 MAPK in 818del4 mutant IFNGR1-expressing cells…………………………………………………………….54
Figure 5………………………………………………………………………………55
Phosphorylation of ERK1/2 was increased in 818del4 mutant IFNGR1-transfected cells after treated IFN-γ……………………………………………………………...56
Figure 6………………………………………………………………………………57
Phosphorylation of p47phox was increased in 818del4 mutant IFNGR1-transfected cells after treated IFN-γ and inhibited by ERK1/2 inhibitor…………………………58

                                    

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校內：2016-08-29公開
校外：2016-08-29公開

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